锦灯笼中酸浆苦素PhysalinF选择性抑制IDH1突变酶的抗肿瘤作用与机制研究

Mutant isocitrate dehydrogenase 1 (IDH1) converts alpha-KG into 2-HG to promote tumorigenesis, and inhibiting mutant IDH1 enzymes becomes a new anti-tumor strategy. At present, there is still a lack of high-efficiency mutant IDH1 inhibitors from natural sources. Our previous studies found that Physalin F, a selective inhibitor of mutant IDH1, had potent anti-proliferation effects on cancer cells, and inhibited the production of 2-HG in cells. We also used gene chip technology to analyze the changes of related genes after Physalin F treatment by genome-wide expression profiling. On this basis, by using IDH1 mutant tumor cells and xenograft tumor models in mice, we intend to evaluate activities of Physalin F in vivo and in vitro. The targeting mutant IDH1 of Physalin F will be further confirmed by molecular biology methods. The structure of Physalin F-IDH1R132H complex will be obtained by protein crystallography. The technology of real-time detection of cell energy metabolism and metabolic flow will be used to study the anti-tumor mechanism of Physalin F by targeting mutant IDH1 in depth. This study will elucidate the molecular pharmacological basis of anti-tumor effects of Physalis alkekengi L. var. franchetii, and provide theoretical and experimental basis for the anti-tumor application of heat-clearing and detoxification drugs of traditional Chinese medicine.

IDH1突变酶将α-KG转变成2-HG而促进肿瘤的生成，抑制IDH1突变酶成为一种新的抗肿瘤策略。目前天然来源的高效IDH1突变酶抑制剂依然缺乏。我们前期研究发现中药锦灯笼中的酸浆苦素Physalin F，是IDH1突变酶的选择性抑制剂，有很好的抗肿瘤细胞增殖作用，在细胞内抑制2-HG的生成；我们也使用了基因芯片技术分析了Physalin F作用后相关基因的改变。在此基础上，本课题拟采用IDH1突变的肿瘤细胞和小鼠移植瘤模型，评价Physalin F的体内外活性，以分子生物学方法进一步确证Physalin F靶向突变酶，以蛋白质晶体学的方法得到Physalin F-IDH1R132H复合物结构，采用细胞能量代谢实时检测、代谢流等技术，深入研究Physalin F靶向IDH1突变酶抗肿瘤的机制。本研究将阐明中药锦灯笼抗肿瘤功效的分子药理学基础，为清热解毒类中药的抗肿瘤应用提供理论和实验基础。

异柠檬酸脱氢酶（isocitrate dehydrogenase，IDH）是参与细胞三羧酸循环的关键限速酶，是目前发现的在肿瘤中基因突变最为频繁的代谢酶，特别是其亚型IDH1的R132位突变更为常见，在多种实体瘤中广泛发生。当前已有针对靶向IDH1R132H突变的小分子天然先导化合物。 天然产物来源于生物体组成物质及其代谢产物，具有结构独特、生物活性广泛、不易耐药等优点，在发掘新颖的抗肿瘤活性成分中起到重要作用。以酶为新的分子作用靶点筛选具有靶向性且具有抗肿瘤活性的天然产物，以其作为先导化合物，而后进行结构改造，已成为研究抗癌药物的一种新思路。 本课题通过重建IDH1R132H重组质粒，大量表达并纯化蛋白质，得到IDH1R132H蛋白的晶体结构；而后对实验室天然产物库中小分子化合物进行酶活性筛选，通过微量热泳动技术评价化合物与IDH1R132H的结合力，用HPLC-MS/MS检测其（R）-2-HG水平，并通过细胞水平实验检测化合物对肿瘤的抑制活性和促凋亡活性，并进行初步的机制验证，发现了一个潜在的IDH1天然小分子抑制剂physalin F，可作为极具潜力的先导化合物，为进一步IDH1抑制剂的研发提供理论和实验基础。并通过蛋白质晶体学、计算生物学和药物化学的方法，对合成的线性非天然肽类似物43等化合物进行抗肿瘤活性评价，采用IDH1突变的脑胶质瘤细胞，进行了较为深入的活性评价和机制究，认为它们是良好的先导化合物，值得进行进一步的临床前研究。